The Src family of non-receptor protein-tyrosine kinases is comprised of eight isozymes in mammalian cells. While c-Src and c-Yes have the highest sequence homology, recent chemical genetics studies from our group have demonstrated opposing roles for these two Src family members in the control of mouse embryonic stem (mES) cell fate. Selective small molecule inhibitors or agonists of c-Src, c-Yes and other SFKs may allow pharmacological manipulation of ES cell growth and differentiation. The development of selective probe compounds that target Src-family kinase ATP-binding sites is challenging due to sequence and structural conservation. Thus, we propose an innovative approach to discover small molecules that target the non-catalytic SH3 and SH2 domains rather than the kinase domain. Our overall goal is to identify selective allosteric modulators for c-Src and c-Yes by taking advantage of differences in the binding preferences of their SH3 and SH2 domains. To achieve this goal, our first Aim is to develop and implement a fluorescence polarization-based high throughput screening assay that is based on recombinant c-Src and c-Yes SH3-SH2-linker proteins and fluorescently labeled peptide probes that selectively bind the SH3 and SH2 domains. Small molecules that bind to and enhance the intramolecular regulatory interactions of the SH3 and/or SH2 domains would likely be potent antagonists of kinase activity. In contrast, small molecules that disrupt these interactions would act as agonists (activators) of the kinases. We anticipate that both classes of small molecules will be detected in our FP- based screening assay. In Aim 2, we will solve the X-ray crystal structures of the c-Src SH3-SH2-linker region and near full-length c-Src in complex with a high-affinity peptide agonist that binds to the SH3 domain. Limited structural information exists for Src-family kinases following activation by SH3 or SH2 domain displacement. Therefore, these structures will provide new insight as to the mechanism of c-Src activation by SH3 domain displacement and will aid in the design of more potent and selective agonists. Our ultimate goal is to identify selective allosteric modulators of Src-family kinase activity that can be used as chemical probes in vivo. In our third Aim, we will test hit compounds from Aim 1 for activity in fibroblast transformation assays in which the transformed phenotype is driven by individual Src-family members. Selective compounds from these studies will then be tested in mES cells, where endogenous c-Src drives differentiation commitment that is opposed by c-Yes. Selective, cell-active allosteric modulators of Src-family members will be valuable probes for individual kinase functions in ES cells and other cell types where multiple family members are expressed and have distinct functions, such as colorectal carcinoma and other forms of cancer. This project will provide the Candidate with a cross- disciplinary training experience in drug discovery, protein biochemistry, structural and chemical biology.